Genome editing has successfully created cell lines and animal models for biological and disease studies, and has a wide range of potential therapeutic applications (Gaj, et al., Trends Biotechnol, 31:397-405 (2013)). In particular, engineered nucleases creating DNA double-strand breaks or single-strand breaks (“nicks”) at specific genomic sequences greatly enhance the rate of genomic manipulation. Double-strand breaks repaired by the cellular non-homologous end joining (NHEJ) pathway often induce insertions, deletions, and mutations, or other events, which are effective for gene disruptions and knockouts. Alternatively, when a donor DNA is supplied, double-strand breaks and DNA nicks can be repaired through homologous recombination, which incorporates the donor DNA and results in precise modification of the genomic sequence. Regardless of the DNA repair pathway, it is important to minimize off-target cleavage in order to reduce the detrimental effects of mutations and chromosomal rearrangements. Although zinc finger nucleases and TAL effector nucleases potentially have a wide range of applications, they were found to cleave at off-target sites at detectable rates (Cornu, et al, Methods Mol Biol, 649:237-245 (2010); Ramirez, et al., Nucleic Acids Res, 40:5560-5568 (2012); Tesson, et al., Nat Biotechnol, 29:695-696 (2011); Hockemeyer, et al., Nat Biotechnol, 29:731-734 (2011); Mussolino, et al., Nucleic Acids Res, 39:9283-9293 (2011)). Clustered regularly interspaced short palindromic repeats (CRISPR), the bacterial defense system using RNA-guided DNA cleaving enzymes (Bolotin, et al., Microbiology, 151 (Pt. 8): 2551-2561 (2005); Barrangou, et al., Science, 315:1709-1712 (2007); Brouns, et al., Science, 321: 960-964 (2008); Hale, et al., Cell, 139: 945-956 (2009); Horvath, et al., Science, 327: 167-170 (2010); Marraffini, et al., Nat Rev Genet, 11:181-190 (2010); Garneau, et al., Nature, 468: 67-71 (2010)) is an exciting alternative to zinc finger nucleases and TAL effector nucleases due to the ease of directing the CRISPR-associated (Cas) proteins (such as Cas9) to multiple gene targets by providing guide RNA sequences complementary to the target sites (Jinek, et al., Science, 337: 816-821 (2012); Cong, et al., Science, 339: 819-823 (2013). Target sites for CRISPR/Cas9 systems can be found near most genomic loci; the only requirement is that the target sequence, matching the guide strand RNA, is followed by a protospacer adjacent motif (PAM) sequence in either orientation (Mojica, et al., Microbiology, 155 (Pt. 3): 733-740 (2009); Shah, et al., RNA Biol, 10:891-899 (2013); Horvath, et al., J Bacteriol, 190:1401-1412 (2008)). For Streptococcus pyogenes (Sp) Cas9, this is any nucleotide followed by a pair of guanines (marked as NGG). Studies on CRISPR/Cas9 systems indicate the possibility of high off-target activity due to nonspecific hybridization of the guide strand to DNA sequences with base pair mismatches at positions distal from the PAM region (Cong, et al., Science, 339: 819-823 (2013); Gasiunas, et al., Proc Natl Acad Sci USA, 109:E2579-E2586 (2012); Jinek, et al., Elife 2:e00471 (2013); Jiang, et al., Nat Biotechnol, 31: 233-239 (2013)).
For CRISPR/Cas9 systems, studies have confirmed levels of off-target cleavage comparable with the on-target rates (Fu, et al., Nat Biotechnol, 31: 822-826 (2013); Hsu, et al., Nat Biotechnol, 31: 827-832 (2013); Cradick, et al., Nucleic Acids Res, 41:9584-9592 (2013); Pattanayak, et al., Nat Biotechnol, 31: 839-843 (2013)), even with multiple mismatches to the guide strand in the region close to the PAM. RNA guide strands containing insertions or deletions in addition to base mismatches can result in cleavage and mutagenesis at genomic target site with levels similar to that of the original guide strand (Lin, et al., Nucleic Acids Res, 42:7473-7485 (2014)). These studies provide the first experimental evidence that genomic sites could be cleaved when the DNA sequences contain insertions or deletions compared with the CRISPR guide strand. These results have demonstrated the need to identify potential off-target sites when choosing guide strand designs and examine off-target effects experimentally when using CRISPR/Cas systems in cells, plants and/or animals.
As mismatches and indels (insertions and deletions) are tolerated between the guide strand and target sequences, there may be embodiments where there are known or unknown differences between the guide stand and its complementary sequences. In some embodiments, the intended mismatches, truncations, indels or other noncomplementary sequences may be included, such that the guide sequence will direct cleavage to the target site, although not a direct matching sequence.
A number of CRISPR tools have been developed, including Cas Online Designer (Hsu, et al., Nat Biotechnol, 31: 827-832 (2013)), ZiFit,27 CRISPR Tools, (Hsu, et al., Nat Biotechnol, 31: 827-832 (2013)) and Cas OFFinder (Bae, et al., Bioinformatics, 30:1473-1475 (2014)), for different functions (Hsu, et al., Nat Biotechnol, 31: 827-832 (2013); Bae, et al., Bioinformatics, 30:1473-1475 (2014); Xiao, et al., Bioinformatics, 30:1180-1182 (2014); Grissa, et al., Nucleic Acids Res, 35: W52-W57 (2007); Grissa, et al., BMC Bioinformatics, 8:172 (2007); Rousseau, et al., Bioinformatics, 25: 3317-3318 (2009); Montague, et al., Nucleic Acids Res, 42:W401-W407 (2014)). However, none of these bioinformatics search tools has considered the off-target sites due to insertions or deletions between target DNA and guide RNA sequences, nor provide application-specific primers. Off-target cleavage could be detected in cells with 15 different insertions and deletions between the guide strand and genomic sequence, sometimes at rates higher than that of the perfectly matched guide strand (Lin, et al., Nucleic Acids Res, 42:7473-7485 (2014)).
Therefore, it is an object of the invention to provide a bioinformatics tool to identify potential off-target sites that have mismatches, insertions, and/or deletions between an RNA guide strand of choice and genomic sequences.
It is a further object of the invention to provide application-specific primers.